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Seth L Alper - One of the best experts on this subject based on the ideXlab platform.
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Molecular cloning and functional characterization of zebrafish Slc4a3/Ae3 anion exchanger
Pflügers Archiv - European Journal of Physiology, 2014Co-Authors: Boris E. Shmukler, Caiyong Chen, Tianxun Zhao, Fabian R. Reimold, John F. Heneghan, Seth L AlperAbstract:The zebrafish genome encodes two slc4a1 genes, one expressed in erythroid tissues and the other in the HR (H^+-ATPase-rich) type of embryonic skin ionocytes, and two SLC4A2 genes, one in proximal pronephric duct and the other in several extrarenal tissues of the embryo. We now report cDNA cloning and functional characterization of zebrafish slc4a3/ae3 gene products. The single ae3 gene on chromosome 9 generates at least two low-abundance ae3 transcripts differing only in their 5'-untranslated regions and encoding a single definitive Ae3 polypeptide of 1170 amino acids. The 7 kb upstream of the apparent initiator Met in ae3 exon 3 comprises multiple diverse, mobile repeat elements which disrupt and appear to truncate the Ae3 N-terminal amino acid sequence that would otherwise align with brain Ae3 of other species. Embryonic ae3 mRNA expression was detected by whole mount in situ hybridization only in fin buds at 24–72 hpf, but was detectable by RT-PCR across a range of embryonic and adult tissues. Epitope-tagged Ae3 polypeptide was expressed at or near the surface of Xenopus oocytes, and mediated low rates of DIDS-sensitive ^36Cl^−/Cl^− exchange in influx and efflux assays. As previously reported for Ae2 polypeptides, ^36Cl^− transport by Ae3 was inhibited by both extracellular and intracellular acidic pH, and stimulated by alkaline pH. However, zebrafish Ae3 differed from Ae2 polypeptides in its insensitivity to NH_4Cl and to hypertonicity. We conclude that multiple repeat elements have disrupted the 5'-end of the zebrafish ae3 gene, associated with N-terminal truncation of the protein and reduced anion transport activity.
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SLC4A2-mediated Cl−/HCO3− exchange activity is essential for calpain-dependent regulation of the actin cytoskeleton in osteoclasts
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Fabienne Coury, Seth L Alper, Gary E. Shull, Serhan Zenger, Andrew K. Stewart, Sebastien Robert Stephens, Lynn Neff, Kelly Tsang, Roland Baron, Antonios O. AliprantisAbstract:Bone remodeling requires osteoclasts to generate and maintain an acidified resorption compartment between the apical membrane and the bone surface to solubilize hydroxyapatite crystals within the bone matrix. This acidification process requires (i) apical proton secretion by a vacuolar H+-ATPase, (ii) actin cytoskeleton reorganization into a podosome belt that forms a gasket to restrict lacunar acid leakage, and (iii) basolateral chloride uptake and bicarbonate extrusion by an anion exchanger to provide Cl− permissive for apical acid secretion while preventing cytoplasmic alkalinization. Here we show that osteoclast-targeted deletion in mice of solute carrier family 4 anion exchanger member 2 (SLC4A2) results in osteopetrosis. We further demonstrate a previously unrecognized consequence of SLC4A2 loss of function in the osteoclast: dysregulation of calpain-dependent podosome disassembly, leading to abnormal actin belt formation, cell spreading, and migration. Rescue of SLC4A2-deficient osteoclasts with functionally defined mutants of SLC4A2 indicates regulation of actin cytoskeletal reorganization by anion-exchange activity and intracellular pH, independent of SLC4A2’s long N-terminal cytoplasmic domain. These data suggest that maintenance of intracellular pH in osteoclasts through anion exchange regulates the actin superstructures required for bone resorption.
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essential for calpain-dependent regulation of the actin cytoskeleton in osteoclasts
2013Co-Authors: Fabienne Coury, Seth L Alper, Gary E. Shull, Serhan Zenger, Andrew K. Stewart, Sebastien Robert Stephens, Lynn Neff, Kelly Tsang, Roland Baron, Antonios O. AliprantisAbstract:per-missive for apical acid secretion while preventing cytoplasmicalkalinization. Here we show that osteoclast-targeted deletion inmice of solute carrier family 4 anion exchanger member 2 (SLC4A2)results in osteopetrosis. We further demonstrate a previously un-recognized consequence of SLC4A2 loss of function in the osteo-clast: dysregulation of calpain-dependent podosome disassembly,leading to abnormal actin belt formation, cell spreading, and mi-gration. Rescue of SLC4A2-deficient osteoclasts with functionallydefined mutants of SLC4A2 indicates regulation of actin cytoskel-etal reorganization by anion-exchange activity and intracellularpH, independent of SLC4A2’s long N-terminal cytoplasmic domain.These data suggest that maintenance of intracellular pH in osteo-clasts through anion exchange regulates the actin superstructuresrequired for bone resorption.
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Molecular physiology and genetics of Na+-independent SLC4 anion exchangers.
Journal of Experimental Biology, 2009Co-Authors: Seth L AlperAbstract:Plasmalemmal Cl–/HCO3– exchangers are encoded by the SLC4 and SLC26 gene superfamilies, and function to regulate intracellular pH, [Cl–] and cell volume. The Cl–/HCO3– exchangers of polarized epithelial cells also contribute to transepithelial secretion and reabsorption of acid–base equivalents and Cl–. This review focuses on Na+-independent electroneutral Cl–/HCO3– exchangers of the SLC4 family. Human SLC4A1/AE1 mutations cause the familial erythroid disorders of spherocytic anemia, stomatocytic anemia and ovalocytosis. A largely discrete set of AE1 mutations causes familial distal renal tubular acidosis. The SLC4A2/Ae2–/– mouse dies before weaning with achlorhydria and osteopetrosis. A hypomorphic Ae2–/– mouse survives to exhibit male infertility with defective spermatogenesis and a syndrome resembling primary biliary cirrhosis. A human SLC4A3/AE3 polymorphism is associated with seizure disorder, and the Ae3–/– mouse has increased seizure susceptibility. The transport mechanism of mammalian SLC4/AE polypeptides is that of electroneutral Cl–/anion exchange, but trout erythroid Ae1 also mediates Cl– conductance. Erythroid Ae1 may mediate the DIDS-sensitive Cl– conductance of mammalian erythrocytes, and, with a single missense mutation, can mediate electrogenic SO42–/Cl– exchange. AE1 trafficking in polarized cells is regulated by phosphorylation and by interaction with other proteins. AE2 exhibits isoform-specific patterns of acute inhibition by acidic intracellular pH and independently by acidic extracellular pH. In contrast, AE2 is activated by hypertonicity and, in a pH-independent manner, by ammonium and by hypertonicity. A growing body of structure–function and interaction data, together with emerging information about physiological function and structure, is advancing our understanding of SLC4 anion exchangers.
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Putative re-entrant loop 1 of AE2 transmembrane domain has a major role in acute regulation of anion exchange by pH.
The Journal of biological chemistry, 2008Co-Authors: Andrew K. Stewart, C. E. Kurschat, Richard D. Vaughan-jones, Seth L AlperAbstract:Normal pH sensitivity of the SLC4A2/AE2 anion exchanger requires transmembrane domain (TMD) amino acid (aa) residues not conserved in the homologous but relatively pH-insensitive SLC4A1/AE1 polypeptide. We tested the hypothesis that the nonconserved aa cluster 1075DKPK1078 within the first putative re-entrant loop (RL1) of AE2 TMD contributes to pH sensor function by studying anion exchange function of AE2 mutants in which these and other RL1 aa were systematically substituted with corresponding RL1 aa from AE1. Regulation of Cl-/Cl- and Cl-/HCO(-)3 exchange by intracellular pH (pHi) or extracellular pH (pHo) was measured as 4,4'-di-isothiocyanatostilbene-2,2' disulfonic acid-sensitive 36Cl- efflux from Xenopus oocytes. AE2 RL1 mutants 1075AAAQ1078 and 1075AAAQN1079 showed reduced pHi sensitivity and pHo sensitivity was acid-shifted by approximately 1 pH unit. Individual mutants D1075A and P1077A exhibited moderately altered pH sensitivity, whereas a range of substitutions at conserved AE2 Ile-1079 substantially altered sensitivity to pHo and/or pHi. Substitution of the complete AE1 RL1 with AE2 RL1 failed to confer AE2-like pH sensitivity onto AE1. Replacement, however, of AE1 RL1 763SGPGAAAQ770 with AE2 1071VAPGDKPK1078 restored pHi sensitivity to the chimera AE2(1-920)/AE1(613-929) without affecting its low sensitivity to pHo. The results show that acute regulation of AE2 by pH requires RL1 of the TMD. We propose that critical segments of RL1 constitute part of an AE2 pH sensor that, together with residues within the N-terminal half of the TMD, constrain the AE2 polypeptide in a conformation required for regulation of anion exchange by pHi.
Timothy Pl Smith - One of the best experts on this subject based on the ideXlab platform.
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A deletion mutation in bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle
BMC Genomics, 2010Co-Authors: Stacey N Meyers, Shannon L Swist, Donal O'toole, Jeffrey R. O'connell, Brandy M. Marron, Jonathan Edward Beever, Tara G. Mcdaneld, David J Steffen, Tad S. Sonstegard, Timothy Pl SmithAbstract:Background Osteopetrosis is a skeletal disorder of humans and animals characterized by the formation of overly dense bones, resulting from a deficiency in the number and/or function of bone-resorbing osteoclast cells. In cattle, osteopetrosis can either be induced during gestation by viral infection of the dam, or inherited as a recessive defect. Genetically affected calves are typically aborted late in gestation, display skull deformities and exhibit a marked reduction of osteoclasts. Although mutations in several genes are associated with osteopetrosis in humans and mice, the genetic basis of the cattle disorder was previously unknown. Results We have conducted a whole-genome association analysis to identify the mutation responsible for inherited osteopetrosis in Red Angus cattle. Analysis of >54,000 SNP genotypes for each of seven affected calves and nine control animals localized the defective gene to the telomeric end of bovine chromosome 4 (BTA4). Homozygosity analysis refined the interval to a 3.4-Mb region containing the SLC4A2 gene, encoding an anion exchanger protein necessary for proper osteoclast function. Examination of SLC4A2 from normal and affected animals revealed a ~2.8-kb deletion mutation in affected calves that encompasses exon 2 and nearly half of exon 3, predicted to prevent normal protein function. Analysis of RNA from a proven heterozygous individual confirmed the presence of transcripts lacking exons 2 and 3, in addition to normal transcripts. Genotyping of additional animals demonstrated complete concordance of the homozygous deletion genotype with the osteopetrosis phenotype. Histological examination of affected tissues revealed scarce, morphologically abnormal osteoclasts displaying evidence of apoptosis. Conclusions These results indicate that a deletion mutation within bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle. Loss of SLC4A2 function appears to induce premature cell death, and likely results in cytoplasmic alkalinization of osteoclasts which, in turn, may disrupt acidification of resorption lacunae.
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a deletion mutation in bovine SLC4A2 is associated with osteopetrosis in red angus cattle
BMC Genomics, 2010Co-Authors: Stacey N Meyers, Shannon L Swist, Donal Otoole, Jeffrey R Oconnell, Brandy M. Marron, Jonathan Edward Beever, Tara G. Mcdaneld, David J Steffen, Tad S. Sonstegard, Timothy Pl SmithAbstract:Osteopetrosis is a skeletal disorder of humans and animals characterized by the formation of overly dense bones, resulting from a deficiency in the number and/or function of bone-resorbing osteoclast cells. In cattle, osteopetrosis can either be induced during gestation by viral infection of the dam, or inherited as a recessive defect. Genetically affected calves are typically aborted late in gestation, display skull deformities and exhibit a marked reduction of osteoclasts. Although mutations in several genes are associated with osteopetrosis in humans and mice, the genetic basis of the cattle disorder was previously unknown. We have conducted a whole-genome association analysis to identify the mutation responsible for inherited osteopetrosis in Red Angus cattle. Analysis of >54,000 SNP genotypes for each of seven affected calves and nine control animals localized the defective gene to the telomeric end of bovine chromosome 4 (BTA4). Homozygosity analysis refined the interval to a 3.4-Mb region containing the SLC4A2 gene, encoding an anion exchanger protein necessary for proper osteoclast function. Examination of SLC4A2 from normal and affected animals revealed a ~2.8-kb deletion mutation in affected calves that encompasses exon 2 and nearly half of exon 3, predicted to prevent normal protein function. Analysis of RNA from a proven heterozygous individual confirmed the presence of transcripts lacking exons 2 and 3, in addition to normal transcripts. Genotyping of additional animals demonstrated complete concordance of the homozygous deletion genotype with the osteopetrosis phenotype. Histological examination of affected tissues revealed scarce, morphologically abnormal osteoclasts displaying evidence of apoptosis. These results indicate that a deletion mutation within bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle. Loss of SLC4A2 function appears to induce premature cell death, and likely results in cytoplasmic alkalinization of osteoclasts which, in turn, may disrupt acidification of resorption lacunae.
Andrew K. Stewart - One of the best experts on this subject based on the ideXlab platform.
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SLC4A2-mediated Cl−/HCO3− exchange activity is essential for calpain-dependent regulation of the actin cytoskeleton in osteoclasts
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Fabienne Coury, Seth L Alper, Gary E. Shull, Serhan Zenger, Andrew K. Stewart, Sebastien Robert Stephens, Lynn Neff, Kelly Tsang, Roland Baron, Antonios O. AliprantisAbstract:Bone remodeling requires osteoclasts to generate and maintain an acidified resorption compartment between the apical membrane and the bone surface to solubilize hydroxyapatite crystals within the bone matrix. This acidification process requires (i) apical proton secretion by a vacuolar H+-ATPase, (ii) actin cytoskeleton reorganization into a podosome belt that forms a gasket to restrict lacunar acid leakage, and (iii) basolateral chloride uptake and bicarbonate extrusion by an anion exchanger to provide Cl− permissive for apical acid secretion while preventing cytoplasmic alkalinization. Here we show that osteoclast-targeted deletion in mice of solute carrier family 4 anion exchanger member 2 (SLC4A2) results in osteopetrosis. We further demonstrate a previously unrecognized consequence of SLC4A2 loss of function in the osteoclast: dysregulation of calpain-dependent podosome disassembly, leading to abnormal actin belt formation, cell spreading, and migration. Rescue of SLC4A2-deficient osteoclasts with functionally defined mutants of SLC4A2 indicates regulation of actin cytoskeletal reorganization by anion-exchange activity and intracellular pH, independent of SLC4A2’s long N-terminal cytoplasmic domain. These data suggest that maintenance of intracellular pH in osteoclasts through anion exchange regulates the actin superstructures required for bone resorption.
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essential for calpain-dependent regulation of the actin cytoskeleton in osteoclasts
2013Co-Authors: Fabienne Coury, Seth L Alper, Gary E. Shull, Serhan Zenger, Andrew K. Stewart, Sebastien Robert Stephens, Lynn Neff, Kelly Tsang, Roland Baron, Antonios O. AliprantisAbstract:per-missive for apical acid secretion while preventing cytoplasmicalkalinization. Here we show that osteoclast-targeted deletion inmice of solute carrier family 4 anion exchanger member 2 (SLC4A2)results in osteopetrosis. We further demonstrate a previously un-recognized consequence of SLC4A2 loss of function in the osteo-clast: dysregulation of calpain-dependent podosome disassembly,leading to abnormal actin belt formation, cell spreading, and mi-gration. Rescue of SLC4A2-deficient osteoclasts with functionallydefined mutants of SLC4A2 indicates regulation of actin cytoskel-etal reorganization by anion-exchange activity and intracellularpH, independent of SLC4A2’s long N-terminal cytoplasmic domain.These data suggest that maintenance of intracellular pH in osteo-clasts through anion exchange regulates the actin superstructuresrequired for bone resorption.
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Putative re-entrant loop 1 of AE2 transmembrane domain has a major role in acute regulation of anion exchange by pH.
The Journal of biological chemistry, 2008Co-Authors: Andrew K. Stewart, C. E. Kurschat, Richard D. Vaughan-jones, Seth L AlperAbstract:Normal pH sensitivity of the SLC4A2/AE2 anion exchanger requires transmembrane domain (TMD) amino acid (aa) residues not conserved in the homologous but relatively pH-insensitive SLC4A1/AE1 polypeptide. We tested the hypothesis that the nonconserved aa cluster 1075DKPK1078 within the first putative re-entrant loop (RL1) of AE2 TMD contributes to pH sensor function by studying anion exchange function of AE2 mutants in which these and other RL1 aa were systematically substituted with corresponding RL1 aa from AE1. Regulation of Cl-/Cl- and Cl-/HCO(-)3 exchange by intracellular pH (pHi) or extracellular pH (pHo) was measured as 4,4'-di-isothiocyanatostilbene-2,2' disulfonic acid-sensitive 36Cl- efflux from Xenopus oocytes. AE2 RL1 mutants 1075AAAQ1078 and 1075AAAQN1079 showed reduced pHi sensitivity and pHo sensitivity was acid-shifted by approximately 1 pH unit. Individual mutants D1075A and P1077A exhibited moderately altered pH sensitivity, whereas a range of substitutions at conserved AE2 Ile-1079 substantially altered sensitivity to pHo and/or pHi. Substitution of the complete AE1 RL1 with AE2 RL1 failed to confer AE2-like pH sensitivity onto AE1. Replacement, however, of AE1 RL1 763SGPGAAAQ770 with AE2 1071VAPGDKPK1078 restored pHi sensitivity to the chimera AE2(1-920)/AE1(613-929) without affecting its low sensitivity to pHo. The results show that acute regulation of AE2 by pH requires RL1 of the TMD. We propose that critical segments of RL1 constitute part of an AE2 pH sensor that, together with residues within the N-terminal half of the TMD, constrain the AE2 polypeptide in a conformation required for regulation of anion exchange by pHi.
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Zebrafish ae2.2 encodes a second SLC4A2 anion exchanger.
American journal of physiology. Regulatory integrative and comparative physiology, 2007Co-Authors: Boris E. Shmukler, C. E. Kurschat, Andrew K. Stewart, Jeffrey S. Clark, Ann Hsu, David H. Vandorpe, Seong-kyu Choe, Yi Zhou, Julio D. Amigo, Barry H. PawAbstract:The genome of zebrafish (Danio rerio) encodes two unlinked genes equally closely related to the SLC4A2/AE2 anion exchanger genes of mammals. One of these is the recently reported zebrafish ae2 gene...
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Acute regulation of mouse AE2 anion exchanger requires isoform-specific amino acid residues from most of the transmembrane domain
The Journal of Physiology, 2007Co-Authors: Andrew K. Stewart, Boris E. Shmukler, C. E. Kurschat, Richard D. Vaughan-jones, Seth L AlperAbstract:The widely expressed anion exchanger polypeptide AE2/SLC4A2 is acutely inhibited by acidic intracellular (pHi), by acidic extracellular pH (pHo), and by the calmodulin inhibitor, calmidazolium, whereas it is acutely activated by NH4+. The homologous erythroid/kidney AE1/SLC4A1 polypeptide is insensitive to these regulators. Each of these AE2 regulatory responses requires the presence of AE2's C-terminal transmembrane domain (TMD). We have now measured 36Cl− efflux from Xenopus oocytes expressing bi- or tripartite AE2–AE1 chimeras to define TMD subregions in which AE2-specific sequences contribute to acute regulation. The chimeric AE polypeptides were all functional at pHo 7.4, with the sole exception of AE2(1-920)/AE1(613-811)/AE2(1120-1237). Reciprocal exchanges of the large third extracellular loops were without effect. AE2 regulation by pHi, pHo and NH4+ was retained after substitution of C-terminal AE2 amino acids 1120–1237 (including the putative second re-entrant loop, two TM spans and the cytoplasmic tail) with the corresponding AE1 sequence. In contrast, the presence of this AE2 C-terminal sequence was both necessary and sufficient for inhibition by calmidazolium. All other tested TMD substitutions abolished AE2 pHi sensitivity, abolished or severely attenuated sensitivity to pHo and removed sensitivity to NH4+. Loss of AE2 pHi sensitivity was not rescued by co-expression of a complementary AE2 sequence within separate full-length chimeras or AE2 subdomains. Thus, normal regulation of AE2 by pH and other ligands requires AE2-specific sequence from most regions of the AE2 TMD, with the exceptions of the third extracellular loop and a short C-terminal sequence. We conclude that the individual TMD amino acid residues previously identified as influencing acute regulation of AE2 exert that influence within a regulatory structure requiring essential contributions from multiple regions of the AE2 TMD.
C. E. Kurschat - One of the best experts on this subject based on the ideXlab platform.
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Putative re-entrant loop 1 of AE2 transmembrane domain has a major role in acute regulation of anion exchange by pH.
The Journal of biological chemistry, 2008Co-Authors: Andrew K. Stewart, C. E. Kurschat, Richard D. Vaughan-jones, Seth L AlperAbstract:Normal pH sensitivity of the SLC4A2/AE2 anion exchanger requires transmembrane domain (TMD) amino acid (aa) residues not conserved in the homologous but relatively pH-insensitive SLC4A1/AE1 polypeptide. We tested the hypothesis that the nonconserved aa cluster 1075DKPK1078 within the first putative re-entrant loop (RL1) of AE2 TMD contributes to pH sensor function by studying anion exchange function of AE2 mutants in which these and other RL1 aa were systematically substituted with corresponding RL1 aa from AE1. Regulation of Cl-/Cl- and Cl-/HCO(-)3 exchange by intracellular pH (pHi) or extracellular pH (pHo) was measured as 4,4'-di-isothiocyanatostilbene-2,2' disulfonic acid-sensitive 36Cl- efflux from Xenopus oocytes. AE2 RL1 mutants 1075AAAQ1078 and 1075AAAQN1079 showed reduced pHi sensitivity and pHo sensitivity was acid-shifted by approximately 1 pH unit. Individual mutants D1075A and P1077A exhibited moderately altered pH sensitivity, whereas a range of substitutions at conserved AE2 Ile-1079 substantially altered sensitivity to pHo and/or pHi. Substitution of the complete AE1 RL1 with AE2 RL1 failed to confer AE2-like pH sensitivity onto AE1. Replacement, however, of AE1 RL1 763SGPGAAAQ770 with AE2 1071VAPGDKPK1078 restored pHi sensitivity to the chimera AE2(1-920)/AE1(613-929) without affecting its low sensitivity to pHo. The results show that acute regulation of AE2 by pH requires RL1 of the TMD. We propose that critical segments of RL1 constitute part of an AE2 pH sensor that, together with residues within the N-terminal half of the TMD, constrain the AE2 polypeptide in a conformation required for regulation of anion exchange by pHi.
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Zebrafish ae2.2 encodes a second SLC4A2 anion exchanger.
American journal of physiology. Regulatory integrative and comparative physiology, 2007Co-Authors: Boris E. Shmukler, C. E. Kurschat, Andrew K. Stewart, Jeffrey S. Clark, Ann Hsu, David H. Vandorpe, Seong-kyu Choe, Yi Zhou, Julio D. Amigo, Barry H. PawAbstract:The genome of zebrafish (Danio rerio) encodes two unlinked genes equally closely related to the SLC4A2/AE2 anion exchanger genes of mammals. One of these is the recently reported zebrafish ae2 gene...
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Acute regulation of mouse AE2 anion exchanger requires isoform-specific amino acid residues from most of the transmembrane domain
The Journal of Physiology, 2007Co-Authors: Andrew K. Stewart, Boris E. Shmukler, C. E. Kurschat, Richard D. Vaughan-jones, Seth L AlperAbstract:The widely expressed anion exchanger polypeptide AE2/SLC4A2 is acutely inhibited by acidic intracellular (pHi), by acidic extracellular pH (pHo), and by the calmodulin inhibitor, calmidazolium, whereas it is acutely activated by NH4+. The homologous erythroid/kidney AE1/SLC4A1 polypeptide is insensitive to these regulators. Each of these AE2 regulatory responses requires the presence of AE2's C-terminal transmembrane domain (TMD). We have now measured 36Cl− efflux from Xenopus oocytes expressing bi- or tripartite AE2–AE1 chimeras to define TMD subregions in which AE2-specific sequences contribute to acute regulation. The chimeric AE polypeptides were all functional at pHo 7.4, with the sole exception of AE2(1-920)/AE1(613-811)/AE2(1120-1237). Reciprocal exchanges of the large third extracellular loops were without effect. AE2 regulation by pHi, pHo and NH4+ was retained after substitution of C-terminal AE2 amino acids 1120–1237 (including the putative second re-entrant loop, two TM spans and the cytoplasmic tail) with the corresponding AE1 sequence. In contrast, the presence of this AE2 C-terminal sequence was both necessary and sufficient for inhibition by calmidazolium. All other tested TMD substitutions abolished AE2 pHi sensitivity, abolished or severely attenuated sensitivity to pHo and removed sensitivity to NH4+. Loss of AE2 pHi sensitivity was not rescued by co-expression of a complementary AE2 sequence within separate full-length chimeras or AE2 subdomains. Thus, normal regulation of AE2 by pH and other ligands requires AE2-specific sequence from most regions of the AE2 TMD, with the exceptions of the third extracellular loop and a short C-terminal sequence. We conclude that the individual TMD amino acid residues previously identified as influencing acute regulation of AE2 exert that influence within a regulatory structure requiring essential contributions from multiple regions of the AE2 TMD.
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Role of nonconserved charged residues of the AE2 transmembrane domain in regulation of anion exchange by pH
Pflügers Archiv - European Journal of Physiology, 2007Co-Authors: A. K. Stewart, C. E. Kurschat, S. L. AlperAbstract:The ubiquitous AE2/SLC4A2 anion exchanger is acutely and independently regulated by intracellular (pH_i) and extracellular pH (pH_o), whereas the closely related AE1/SLC4A1 of the red cell and renal intercalated cell is relatively pH-insensitive. We have investigated the contribution of nonconserved charged residues within the C-terminal transmembrane domain (TMD) of AE2 to regulation by pH through mutation to the corresponding AE1 residues. AE2-mediated Cl^−/Cl^− exchange was measured as 4,4′-di-isothiocyanatostilbene-2,2′-disulfonic acid-sensitive ^36Cl^− efflux from Xenopus oocytes by varying pH_i at constant pH_o, and by varying pH_o at near-constant pH_i. All mutations of nonconserved charged residues of the AE2 TMD yielded functional protein, but mutations of some conserved charged residues (R789E, R1056A, R1134C) reduced or abolished function. Individual mutation of AE2 TMD residues R921, F922, P1077, and R1107 exhibited reduced pH_i sensitivity compared to wt AE2, whereas TMD mutants K1153R, R1155K, R1202L displayed enhanced sensitivity to acidic pH_i. In addition, pH_o sensitivity was significantly acid- shifted when nonconserved AE2 TMD residues E981, K982, and D1075 were individually converted to the corresponding AE1 residues. These results demonstrate that multiple conserved charged residues are important for basal transport function of AE2 and that certain nonconserved charged residues of the AE2 TMD are essential for wild-type regulation of anion exchange by pH_i and pH_o.
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Transmembrane domain histidines contribute to regulation of AE2-mediated anion exchange by pH.
American journal of physiology. Cell physiology, 2006Co-Authors: Andrew K. Stewart, C. E. Kurschat, Richard D. Vaughan-jones, Daniel Burns, Nurindura Banger, Seth L AlperAbstract:Activity of the AE2/SLC4A2 anion exchanger is modulated acutely by pH, influencing the transporter's role in regulation of intracellular pH (pHi) and epithelial solute transport. In Xenopus oocytes...
Stacey N Meyers - One of the best experts on this subject based on the ideXlab platform.
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A deletion mutation in bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle
BMC Genomics, 2010Co-Authors: Stacey N Meyers, Shannon L Swist, Donal O'toole, Jeffrey R. O'connell, Brandy M. Marron, Jonathan Edward Beever, Tara G. Mcdaneld, David J Steffen, Tad S. Sonstegard, Timothy Pl SmithAbstract:Background Osteopetrosis is a skeletal disorder of humans and animals characterized by the formation of overly dense bones, resulting from a deficiency in the number and/or function of bone-resorbing osteoclast cells. In cattle, osteopetrosis can either be induced during gestation by viral infection of the dam, or inherited as a recessive defect. Genetically affected calves are typically aborted late in gestation, display skull deformities and exhibit a marked reduction of osteoclasts. Although mutations in several genes are associated with osteopetrosis in humans and mice, the genetic basis of the cattle disorder was previously unknown. Results We have conducted a whole-genome association analysis to identify the mutation responsible for inherited osteopetrosis in Red Angus cattle. Analysis of >54,000 SNP genotypes for each of seven affected calves and nine control animals localized the defective gene to the telomeric end of bovine chromosome 4 (BTA4). Homozygosity analysis refined the interval to a 3.4-Mb region containing the SLC4A2 gene, encoding an anion exchanger protein necessary for proper osteoclast function. Examination of SLC4A2 from normal and affected animals revealed a ~2.8-kb deletion mutation in affected calves that encompasses exon 2 and nearly half of exon 3, predicted to prevent normal protein function. Analysis of RNA from a proven heterozygous individual confirmed the presence of transcripts lacking exons 2 and 3, in addition to normal transcripts. Genotyping of additional animals demonstrated complete concordance of the homozygous deletion genotype with the osteopetrosis phenotype. Histological examination of affected tissues revealed scarce, morphologically abnormal osteoclasts displaying evidence of apoptosis. Conclusions These results indicate that a deletion mutation within bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle. Loss of SLC4A2 function appears to induce premature cell death, and likely results in cytoplasmic alkalinization of osteoclasts which, in turn, may disrupt acidification of resorption lacunae.
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a deletion mutation in bovine SLC4A2 is associated with osteopetrosis in red angus cattle
BMC Genomics, 2010Co-Authors: Stacey N Meyers, Shannon L Swist, Donal Otoole, Jeffrey R Oconnell, Brandy M. Marron, Jonathan Edward Beever, Tara G. Mcdaneld, David J Steffen, Tad S. Sonstegard, Timothy Pl SmithAbstract:Osteopetrosis is a skeletal disorder of humans and animals characterized by the formation of overly dense bones, resulting from a deficiency in the number and/or function of bone-resorbing osteoclast cells. In cattle, osteopetrosis can either be induced during gestation by viral infection of the dam, or inherited as a recessive defect. Genetically affected calves are typically aborted late in gestation, display skull deformities and exhibit a marked reduction of osteoclasts. Although mutations in several genes are associated with osteopetrosis in humans and mice, the genetic basis of the cattle disorder was previously unknown. We have conducted a whole-genome association analysis to identify the mutation responsible for inherited osteopetrosis in Red Angus cattle. Analysis of >54,000 SNP genotypes for each of seven affected calves and nine control animals localized the defective gene to the telomeric end of bovine chromosome 4 (BTA4). Homozygosity analysis refined the interval to a 3.4-Mb region containing the SLC4A2 gene, encoding an anion exchanger protein necessary for proper osteoclast function. Examination of SLC4A2 from normal and affected animals revealed a ~2.8-kb deletion mutation in affected calves that encompasses exon 2 and nearly half of exon 3, predicted to prevent normal protein function. Analysis of RNA from a proven heterozygous individual confirmed the presence of transcripts lacking exons 2 and 3, in addition to normal transcripts. Genotyping of additional animals demonstrated complete concordance of the homozygous deletion genotype with the osteopetrosis phenotype. Histological examination of affected tissues revealed scarce, morphologically abnormal osteoclasts displaying evidence of apoptosis. These results indicate that a deletion mutation within bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle. Loss of SLC4A2 function appears to induce premature cell death, and likely results in cytoplasmic alkalinization of osteoclasts which, in turn, may disrupt acidification of resorption lacunae.